Gap junction remodeling is one of the common pathological bases involved in the onset and development of diseases such as arrhythmia, tumor, atherosclerosis, and the like, whereas connexin 43 (Cx43) is the basic structure unit constituting the gap junction as well as the main structure basis constituting gap junctions between myocardial cells. Cx43 is a type of connexin having a molecular weight of 43 Kd, the main role of which is to mediate the direct communication between adjacent cells. In heart, it mainly functions to form fast electrical impulses between cells, ensure the synchronism and harmony of the overall electrical activities in the heart, and maintain the electrical activities of myocardial cells and the synchronism of mechanical functions of contraction and relaxation. Consequently, the variation of Cx43 in terms of the amount, distribution, function, phosphorylation state, etc. (i.e., gap junction remodeling) will definitely result in electrical coupling dysfunction between myocardial cells, which mainly manifests as change of synchronism and harmony of electrical activities between cells, slowing down of electrical conduction, change of aeolotropy, etc. Various degrees of Cx43 gap junction remodeling occur in various adult acquired cardiovascular diseases, such as myocardial ischemia, arrhythmia, cardiac failure, hypertension, atherosclerosis, etc. Cx43 gap junction remodeling is an important structural basis for generation of various arrhythmia, especially reentrant tachycardia, and is also the primary cause for death and sudden death from various heart diseases.
Meanwhile, Cx43 is involved in various processes from growth to death in many cellular life cycles, directly mediates transmission of cellular growth regulatory signals between adjacent cells, and regulates cell growth, differentiation and apoptosis. The abnormity of Cx43 will directly result in abnormity of cellular growth regulation and disappearance of contact inhibition, terminal differentiation and apoptosis ability, which are manifested as extended or immortalized cell life cycle, and thereby leading to tumorigenesis.
Therefore, Cx43 has become a new research hotspot in the field of researches on cardiovascular diseases and tumor at home and abroad as a common molecule target for antiarrhythmic treatment and antitumor treatment. Many in vitro research methods are also developed accordingly for analysis of the amount and function (including phosphorylation state) of Cx43. These research methods include: applying PCR or RT-PCR to detect the expression of Cx43 at mRNA level; applying Western Blot to carry out semi-quantification studies on Cx43 proteins; observing subcellular level localization, gap junction remodeling, etc. of Cx43 through immunohistochemical and immunofluorescent staining techniques; analyzing the phosphorylation state of different phosphorylation sites of Cx43 by introducing specific antibodies of different phosphorylation sites of Cx43 and using matrix assisted laser desorption/ionization mass spectrometry, immobilized metal affinity chromatography, and LC-MS; using intercellular transfer assay of fluorescent dyes, introducing small molecular fluorescent dyes into cytoplasm through micro-injection method, in combination with fluorescence microscope technique, to assess the direct intercellular communication function mediated by Cx43 at cellular level; and the like. However, these in vitro detection processes mainly rely on analysis of vast results of experiments at cellular level or on ex vivo tissues.
Although in vitro detection processes can be used to judge and analyze Cx43 expression level and functional status of cells or tissues, all of them have certain limitations at the technical level:
(1) They require to obtain samples via cell culture, biopsy or autopsy, and therefore cannot be applied to human body directly;
(2) in vitro experimental results may not comply with the true situation in vivo: experimental conditions, devices and methods have great influence on in vitro tests, and some important ingredients in the sample may be lost during treatment thereof, leading to larger error, which makes the experimental results not comply with the true situation in vivo;
(3) in vitro experiments go against dynamic studies: in vitro experiments require to execute experimental animals at different time points to acquire tissues or obtain materials through repeated biopsies, can only be used to observe certain stage of a disease, cannot achieve the actual dynamic research in the same animal, and are difficult to come into an accurate conclusion on the whole process of such a complex, progressive disease;
(4) Their operations are complicated, time-consuming and costly, and are substantially stochastic. Consequently, in vivo visual research of Cx43 can provide a new idea for solving these problems, whereas the appearance of molecular imaging technology makes it become possible.
Molecular imaging is a subject for carrying out imaging, qualitative and quantitative researches on biological processes in living human and animal body at cellular and molecular levels through imaging methods. It is notably characterized by application of a molecular probe, and employs multiple imaging means to image specific target in vivo. Imaging means include radionuclide imaging, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MR spectroscopy, MRS), optical imaging (OI), ultrasound imaging (US) and integration of multi-mode imaging, etc. By means of these imaging techniques, certain specific physiological or pathological processes in a life system, such as gene expression, interaction between proteins, signal transduction, metabolism of cells, cell tracking, etc., can become visual in the form of images.
In the Cx43 visualization protocol performed by V Baklaushev et al., a probe is synthesized using the specific antibody of Cx43 protein extracellular E2 segment as a targeted affinity component, and using radioactive isotope 125I and fluorescent dye Alexa660 as signal components. a γ-ray counter and a fluorescence microscope are utilized to detect the abnormal expression of Cx43 in brain glioma, but still failing to get rid of the limitations of in vitro detection methods:
(1) 125I is not a nuclein suitable for in vivo imaging, only the γ-ray counter can be used for detection, and the test results are not visual enough. Therefore, in the study, after intravenous injection of the probe 125I-MAbE2Cx43, the animals were sacrificed and tissues were acquired, and a γ-ray counter was employed to carry out radioactivity analysis;
(2) Alexa660 is also not a fluorescent dye suitable for in vivo imaging, can only be used in an immunohistochemical staining analysis, and therefore, in the study, after intravenous injection of the probe Alexa660-MAbE2Cx43, the animals were sacrificed and tissues were acquired and produced into a frozen section, and a fluorescence microscope was employed to analyze the tissues;
(3) the MAbE2Cx43 antibody used as the affinity component is expensive, can induce immunogenic response, and thereby induce side effects. What's more, due to the larger molecular weight of the antibody, the non-specific absorption will be higher, and it is difficult to obtain ideal pharmacokinetic characteristics and biological distribution characteristics after injection into the body, which renders the detection results inaccurate, and the method inconvenient to apply. Currently, there exists an urgent need for a process for in vivo detection and quantification of Cx43.